Abstract 2890: Anti-tumor effect and mechanism of action of a nuclear transportable antibody drug conjugate Y-TR-1: Anti CD26 antibody conjugated with RNA polymerase II inhibitor triptolide

Abstract Introduction: CD26 is a multifunctional transmembrane glycoprotein that is overexpressed in many cancers. We established the humanized anti-CD26 monoclonal antibody YS110, which has a direct anti-tumor effect through nuclear translocation of CD26 and transcriptional suppression of the POLR2A gene, a subunit of RNA polymerase II (Pol II). Subsequently, we developed an antibody-drug conjugate Y-TR-1 with YS110 and the Pol II inhibitor triptolide and evaluated its in vitro and in vivo antitumor efficacy and toxicity. We then elucidated the mechanism of action of Y-TR-1. Experimental procedures: 5 triptolide molecules on average were crosslinked to YS110 by the heterobifunctional linker SMCC (designated Y-TR-1). The in vitro cytotoxicity of Y-TR-1 was analyzed in CD26-positive or -negative cancer cells and normal cells. The in vivo antitumor effect of Y-TR-1 was assessed using a xenograft assay. Pharmacokinetics/pharmacodynamics studies and toxicity tests were conducted using cynomolgus monkeys. Western blotting and immunofluorescence imaging were performed to confirm the nuclear translocation of Y-TR-1. The inhibitory effect of Y-TR-1 against Pol II was analyzed using quantitative polymerase chain reaction and a live cell imaging assay. The molecular mechanism of Y-TR-1 nuclear translocation was elucidated by immunofluorescence imaging. Results: A conjugation method of Y-TR-1 was established, and the average drug-to-antibody ratio (DAR) was 4.76 by LC/MS. Y-TR-1 showed in vitro cytotoxicity on CD26-positive cancer cells, whereas non-cancer cells were less susceptible to Y-TR-1. The in vivo antitumor effect of Y-TR-1 was confirmed in a xenograft study using three CD26-positive cancer cell lines. Toxicity studies of cynomolgus monkeys with cross-reactivity to Y-TR-1 revealed no severe toxicity. The animals that received 30 mg/kg body weight of Y-TR-1 showed some decline in the reticulocyte count and slight increase in the thrombocyte count, whereas no histological damage was observed in the liver, kidney, heart, or lungs. Nuclear translocated Y-TR-1 was confirmed in the nuclear fraction of CD26-positive cancer cells by western blotting or immunofluorescence imaging. Quantitative polymerase chain reaction and live cell imaging assays revealed Pol II inhibition by Y-TR-1. Y-TR-1 was internalized via caveolae-dependent endocytosis and subsequently colocalized with endosomes before translocation to the nucleus. Conclusions: Y-TR-1 is an ADC with a novel mechanism of action, i.e., nuclear translocation and subsequent Pol II inhibition. Transcriptional repression of POLR2A expression by nuclear-localized CD26 and Pol II inhibition by internalized triptolide are expected to have additive effects. Y-TR1 exhibited potent antitumor activity against CD26-positive cancer cells in vitro and in vivo. Citation Format: Mutsumi Hayashi, Hiroko Madokoro, Koji Yamada, Ryo Hatano, Chikao Morimoto, Taketo Yamada. Anti-tumor effect and mechanism of action of a nuclear transportable antibody drug conjugate Y-TR-1: Anti CD26 antibody conjugated with RNA polymerase II inhibitor triptolide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2890.